Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.

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